Compositions and methods for identifying Ehrlichia species

ABSTRACT

The invention provides methods, kits, compositions, and devices useful for detection of antibodies that bind to  Ehrlichia  antigens and/or for differentiation of certain  Ehrlichia  species from others. In particular, the invention provides methods and kits useful for identifying species of  Ehrlichia  using populations of isolated peptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/252,696, filed Apr. 14, 2014, which claims the benefit of U.S.Provisional Application No. 61/975,581, filed Apr. 4, 2014, which ishereby incorporated by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:ABAX_043_01US_SeqList_ST25.txt, date recorded Apr. 10, 2014, file size86 kilobytes).

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods fordetecting bacterial infection and identifying bacteria species. Inparticular, the invention relates to peptide compositions, methods, andkits for detecting antibodies against bacterial antigens (e.g. antigensfrom Ehrlichia spp.).

BACKGROUND OF THE INVENTION

Ehrlichia bacteria are obligate intracellular pathogens that infectcirculating lymphocytes in mammalian hosts. The most natural mode ofEhrlichia transmission is via a variety of tick vectors. Ehrlichia canis(E. canis) and Ehrlichia chaffeensis (E. chaffeensis) are members of thesame sub-genus group of Ehrlichia that infect canines and humans andcause canine monocytic ehrlichiosis (CME) and human monocyticehrlichiosis (HME), respectively. Another species of Ehrlichia known asEhrlichia ewingii (E. ewingii) has tropism for granulocytes and causesgranulocytic ehrlichiosis. The canine disease is characterized by fever,epilepsy, incoordination, lethargy, bleeding episodes, lymphadenopathy,weight loss, and pancytopenia. In humans the disease is characterized byfever, headache, myalgia, and leukopenia.

Indirect immunofluorescence assays (IFA) and enzyme-linked immunosorbentassays (ELISA) have typically been used in the diagnosis of thesediseases. These assays measure or otherwise detect the binding ofanti-Ehrlichia antibodies from a subject's blood, plasma, or serum toinfected cells, cell lysates, or partially purified whole Ehrlichiaproteins. However, currently known assays for detecting anti-Ehrlichiaantibodies or fragments thereof are severely limited in usefulnessbecause of sensitivity and specificity issues directly related to theimpure nature of the Ehrlichia antigen(s) used in these tests.

The diseases caused by bacteria belonging to different Ehrlichia speciesmanifest differently and require separate management routine (Thomas, R.J., et al.; Expert Rev Anti Infect Ther. 2009 August; 7(6): 709-722). Itis, therefore, important to identify the Ehrlichia species that causes aparticular infection. The currently known immunoassays use mixtures ofmany whole Ehrlichia antigens or antigens that are not species specific.PCR methods, which may be useful to identify Ehrlichia species, areuseable only if the tick is recovered and/or the tissue from host istested soon after infection. Furthermore, cultivation of bacteria fromthe infection site, another method which may be useful to identifyEhrlichia species, is not only technically complex but also requiresfreshly infected tissue. In addition, a cultivation method for thespecies E. ewingii has not yet been developed.

Accordingly, there remains a need in the art for additional assays fordetecting Ehrlichia antigens and serodiagnosis of monocytic ehrlichiosisand granulocytic ehrlichiosis. In particular, there remains a need foran assay for identifying Ehrlichia species, especially an assay that canbe used in a variety of circumstances and for various samples, includingsamples that do not require isolation from freshly infected tissues. Thepresent invention provides methods, compositions, and kits to facilitatethe diagnosis, the species identification, and the treatment of thevarious types of Ehrlichia infections.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery thatparticular mixtures, or populations, of Ehrlichia peptides or theirvariants have preferential binding affinity for antibodies elicited byantigens from particular Ehrlichia species. The inventors have foundthat a particular combination of these peptide mixtures or populationscan be used to identify the Ehrlichia species inducing the antibodyresponse. Accordingly, the present invention provides a method foridentifying the species of Ehrlichia infecting a subject.

In certain embodiments, the method for identifying the species ofEhrlichia infecting a subject comprises:

-   -   contacting a sample from the subject with a first population of        isolated peptides;    -   detecting formation of a first set of complexes comprising an        antibody and one or more peptides in the first population;    -   contacting said sample with a second population of isolated        peptides; and    -   detecting formation of a second set of complexes comprising an        antibody and one or more peptides in the second population,        wherein formation of both the first and second sets of complexes        indicates that the subject is infected with E. ewingii, and        wherein formation of the first but not the second set of        complexes indicates that the subject is infected with E. canis        and/or E. chaffeensis. In some embodiments, the first population        of isolated peptides comprises at least three different        peptides, each comprising a sequence of        S-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-X₃₉-A-E-T-R-X₄₄-T-F-G-L-X₄₉-K-Q-Y-D-G-A-X₅₆-I-X₅₈-E-N-Q-V-Q-N-K-F-T-I-S-N-C        (SEQ ID NO: 1) or a fragment thereof, wherein X₂ is an amino        acid selected from the group consisting of A and V, X₅ is an        amino acid selected from the group consisting of E and D, X₈ is        an amino acid selected from the group consisting of T and P, X₁₀        is an amino acid selected from the group consisting of T and V,        X₁₁ is an amino acid selected from the group consisting of G and        A, X₁₂ is an amino acid selected from the group consisting of L        and V, X₁₃ is an amino acid selected from the group consisting        of Y and F, X₁₈ is an amino acid selected from the group        consisting of D and N, X₂₀ is an amino acid selected from the        group consisting of D and N, X₂₂ is an amino acid selected from        the group consisting of S and V, X₂₃ is an amino acid selected        from the group consisting of A, S, and T, X₂₄ is an amino acid        selected from the group consisting of A and I, X₂₅ is an amino        acid selected from the group consisting of T and P, X₂₆ is an        amino acid selected from the group consisting of S, N, and K,        X₃₉ is any amino acid, X₄₄ is any amino acid, X₄₉ is any amino        acid, X₅₆ is any amino acid, and X₅₈ is any amino acid. In        related embodiments, the second population of isolated peptides        comprises at least three different peptides, each comprising a        sequence of        F-S-A-K-E-E-X₇-A-E-T-R-X₁₂-T-F-G-L-X₁₇-K-Q-Y-D-G-A-X₂₄-I-X₂₆-E-N-Q-V-Q-N-K-F-T-I-S-N-C        (SEQ ID NO: 2) or a fragment thereof, wherein X₇ is any amino        acid, X₁₂ is any amino acid, X₁₇ is any amino acid, X₂₄ is any        amino acid, and X₂₆ is any amino acid.

In certain other embodiments, the method comprises:

-   -   contacting a sample from the subject with a first population of        isolated peptides as described herein;    -   detecting formation of a first set of complexes comprising an        antibody and one or more peptides in the first population;    -   contacting said sample with a third population of isolated        peptides; and    -   detecting formation of a third set of complexes comprising an        antibody and one or more peptides in the third population,        wherein formation of both the first and third sets of        antibody-peptide complexes indicates that the subject is        infected with E. canis and/or E. chaffeensis, and wherein        formation of the first but not the third set of antibody-peptide        complexes indicates that the subject is infected with E.        ewingii. In certain embodiments, the third population of        isolated peptides comprises at least three different peptides,        each comprising a sequence of        S-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-X₃₉-A-X₄₁-T-R-X₄₄-T-F-G-X₄₈-X₄₉-K-Q-Y-D-G-A-X₅₆-I-X₅₈-E-N-Q-V-Q-N-K-F-T-I-S-N-C        (SEQ ID NO: 3) or a fragment thereof, wherein X₂ is an amino        acid selected from the group consisting of A and V, X₅ is an        amino acid selected from the group consisting of E and D, X₈ is        an amino acid selected from the group consisting of T and P, X₁₀        is an amino acid selected from the group consisting of T and V,        X₁₁ is an amino acid selected from the group consisting of G and        A, X₁₂ is an amino acid selected from the group consisting of L        and V, X₁₃ is an amino acid selected from the group consisting        of Y and F, X₁₈ is an amino acid selected from the group        consisting of D and N, X₂₀ is an amino acid selected from the        group consisting of D and N, X₂₂ is an amino acid selected from        the group consisting of S and V, X₂₃ is an amino acid selected        from the group consisting of A, S, and T, X₂₄ is an amino acid        selected from the group consisting of A and I, X₂₅ is an amino        acid selected from the group consisting of T and P, X₂₆ is an        amino acid selected from the group consisting of S, N, and K,        X₃₉ is any amino acid, X₄₁ is an amino acid selected from the        group consisting of D and N, X₄₄ is any amino acid, X₄₈ is an        amino acid selected from the group consisting of V and A, X₄₉ is        any amino acid, X₅₆ is any amino acid, and X₅₈ is any amino        acid.

In certain embodiments, the method comprises:

-   -   contacting a sample from the subject with a first population of        isolated peptides as described herein;    -   detecting formation of a first set of complexes comprising an        antibody and one or more peptides in the first population;    -   contacting said sample with a second population of isolated        peptides as described herein; detecting formation of a second        set of complexes comprising an antibody and one or more peptides        in the second population;    -   contacting said sample with a third population of isolated        peptides as described herein; and    -   detecting formation of a third set of complexes comprising an        antibody and one or more peptides in the third population,        wherein formation of both the first and second sets of complexes        but not the third set indicates that the subject is infected        with E. ewingii, and wherein formation of both the first and        third sets of complexes but not the second set indicates that        the subject is infected with E. canis and/or E. chaffeensis.

In some embodiments of the methods, one or more peptides in the firstpopulation of peptides comprises a fragment of SEQ ID NO: 1. Thefragment of SEQ ID NO: 1 may comprise at least 20, 25, 30, 35, or 40contiguous amino acids from SEQ ID NO: 1. In certain embodiments, thefragment of SEQ ID NO: 1 comprises amino acids 33 to 71 of SEQ ID NO: 1.In particular embodiments, each peptide in the first populationcomprises a sequence of SEQ ID NO: 1.

In certain other embodiments of the methods, one or more peptides in thesecond population of peptides comprises a fragment of SEQ ID NO: 2. Thefragment of SEQ ID NO: 2 may comprise at least 15, 20, 25, 30, or 35contiguous amino acids from SEQ ID NO: 2. In some embodiments, eachpeptide in the second population comprises a sequence of SEQ ID NO: 2.

In other embodiments of the methods, one or more peptides in the thirdpopulation of peptides comprises a fragment of SEQ ID NO: 3. Thefragment of SEQ ID NO: 3 may comprise at least 20, 25, 30, 35, or 40contiguous amino acids from SEQ ID NO: 3. In certain embodiments, thefragment of SEQ ID NO: 3 comprises amino acids 33 to 71 of SEQ ID NO: 3.In particular embodiments, each peptide in the third populationcomprises a sequence of SEQ ID NO: 3.

In some embodiments of the methods, the sample is further analyzed withat least one assay to determine whether the infecting species is E.canis or E. chaffeensis.

In certain embodiments, at least one of the detecting steps in any ofmethods described herein may comprise: (i) performing an ELISA assay;(ii) running a lateral flow assay; (iii) performing an agglutinationassay; (iv) performing a Western blot, slot blot, or dot blot assay; (v)performing a wavelength shift assay; (vi) running the sample through ananalytical or centrifugal rotor; or (vii) running a microarray assay. Insome embodiments, one or more of the detecting steps comprises spinningthe sample in an analytical or centrifugal rotor. In other embodiments,one or more of the detecting steps comprises analyzing the sample withan electrochemical sensor, an optical sensor, chemiluminescence sensoror an opto-electronic sensor. In particular embodiments, one or more ofthe detecting steps comprises performing an ELISA assay or a lateralflow assay.

Certain embodiments of the method further comprise reporting detectionresults. The reporting can be done electronically, in writing, orverbally. It can be done via a machine such as a computer.

In another aspect, the invention provides kits for detecting antibodiesthat bind to Ehrlichia antigens and/or identifying the species ofEhrlichia infecting a subject. In certain embodiments, the kit comprisesone, two, or three different populations of peptides of the invention asdescribed herein. In certain embodiments, the kits further comprise aninstruction for using the peptide populations to identify the species ofEhrlichia in a biological sample. In some embodiments, the kit furthercomprises one or more labeling reagents.

In certain embodiments of the methods or the kits of inventions, thepeptides in the populations of isolated peptides are attached to orimmobilized on a solid support.

Additional aspects and embodiments of the invention will be apparentfrom the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of an embodiment of a method for identifyingEhrlichia species. The abbreviation “EAL” represents ELISA score for anELISA assay using peptide population EE13 (SEQ ID NO: 2), while “CAL”represents ELISA score for an ELISA assay using peptide populationEE12EW1 (SEQ ID NO: 3). In this embodiment, a whole blood sample istested in an ELISA assay, ELISA ECHEW1, using peptide population ECHEW1(SEQ ID NO: 1), which comprises a first population of peptides asdescribed herein. If the result of ELISA ECHEW1 is positive, the samplethen undergoes another ELISA assay, ELISA EE13, using peptide populationEE13, which comprises a second population of peptides as describedherein, and undergoes yet another ELISA assay, ELISA EE12EW, usingpeptide population EE12EW, which comprises a third population ofpeptides as described herein. A positive result of ELISA EE13 combinedwith negative result of ELISA EE12EW, or a higher EAL than CAL,indicates that the sample is infected with E. ewingii. A positive resultof ELISA EE12EW combined with negative result of ELISA EE13, or a higherCAL than EAL, indicates that the sample is infected with E. canis and/orE. chaffeensis. If the sample is identified to be infected with E. canisand/or E. chaffeensis, the sample then undergoes another assay, in thisexample an IFA assay for E. canis or E. chaffeensis, concurrently ornon-concurrently, to determine whether the sample is infected with E.canis or E. chaffeensis.

FIG. 2 is a graphical representation of anti-Ehrlichia antibody scoresof plasma samples drawn at various times from dogs infected with theindicated Ehrlichia species. Dogs were experimentally infected withvarious species of Ehrlichia, and plasma samples were drawn on variousdays post infection as indicated in the graphs. ELISA assays wereperformed on the samples using each of the three populations ofpeptides, ECHEW1, EE12EW, and EE13. The top left and bottom left panelsshow the results from samples separately taken from two dogs infectedwith E. canis. The top right panel shows the results from samples takenfrom a dog infected with E. chaffeensis. Antibody scores were calculatedusing methods described herein.

DETAILED DESCRIPTION

As used herein, the following terms shall have the following meanings:

The term “antigen,” as used herein, refers to a molecule capable ofbeing recognized by an antibody. An antigen can be, for example, apeptide or a modified form thereof. An antigen can comprise one or moreepitopes.

The term “epitope,” as used herein, is a portion of an antigen that isspecifically recognized by an antibody. An epitope, for example, cancomprise or consist of a portion of a peptide (e.g., a peptide of theinvention). An epitope can be a linear epitope, sequential epitope, or aconformational epitope. In certain embodiments, epitopes may comprisenon-contiguous regions.

The term “OMP-1 protein” refers to any of the Outer Membrane Protein 1paralogs of Ehrlichia, including, but not limited to, E. canis P-30, E.canis P30-1, E. chaffeensis P28, E. chaffeensis OMP-1C, E. chaffeensisOMP-1D, E. chaffeensis OMP-1E, and E. chaffeensis OMP-1F.

The term “MSP4 protein” refers to any member of the Surface Antigen MSP4family of Ehrlichia, including, but not limited to, E. canis MSP4,P30-5, and P28-1. OMP and MSP are allelic variants.

The terms “nucleic acid,” “oligonucleotide” and “polynucleotide” areused interchangeably herein and encompass DNA, RNA, cDNA, whether singlestranded or double stranded, as well as chemical modifications thereof.

Single letter amino acid abbreviations used herein have their standardmeaning in the art, and all peptide sequences described herein arewritten according to convention, with the N-terminal end to the left andthe C-terminal end to the right.

The term “score” as used herein refers to a relative value, level,strength, or degree of an assay result. It can be artificially createdby a person of skill in the art or by using an algorithm, sometimesusing samples with known analytes, e.g., antigens or antibodies,optionally using samples with known concentrations or titers of theknown analytes. It can be a number assigned manually by a person ofskill in the art or generated with a formula or algorithm. It can alsobe a symbol, e.g., “−”, “+”, or “++”. A score can be generated fromcalculation with a formula or algorithm, or can be assigned by visualinspection, measurement, or estimation of the assay result. When usingsamples with known concentrations or titers of known analytes, suchsamples can be assayed in diluted and undiluted conditions, and a rangeof scores or a standard curve of scores can be generated, which can beused to assign or estimate the scores of unknown samples assayed for thesame analytes, preferably with the same assays.

Additional terms shall be defined, as required, in the detaileddescription that follows.

The present invention is based, in part, on the discovery thatparticular mixtures, or populations, of Ehrlichia peptides or theirvariants have preferential binding affinity for antibodies elicited byantigens from particular Ehrlichia species. The inventors have foundthat a particular combination of these peptide mixtures or populationscan be used to identify the Ehrlichia species inducing the antibodyresponse. Accordingly, the present invention provides a method foridentifying the species of Ehrlichia infecting a subject, if present.

In certain embodiments, the method for identifying the species ofEhrlichia infecting a subject, if present, comprises:

-   -   contacting a sample from the subject with a first population of        isolated peptides as described herein;    -   detecting formation of a first set of complexes comprising an        antibody and one or more peptides in the first population;    -   contacting said sample with a second population of isolated        peptides as described herein; and    -   detecting formation of a second set of complexes comprising an        antibody and one or more peptides in the second population,        wherein formation of both the first and second sets of complexes        indicates that the subject is infected with E. ewingii, and        wherein formation of the first but not the second set of        complexes indicates that the subject is infected with E. canis        and/or E. chaffeensis.

In other embodiments, the method for identifying the species ofEhrlichia infecting a subject, if present, comprises:

-   -   contacting a sample from the subject with a first population of        isolated peptides as described herein;    -   detecting formation of a first set of complexes comprising an        antibody and one or more peptides in the first population;    -   contacting said sample with a third population of isolated        peptides as described herein; and    -   detecting formation of a third set of complexes comprising an        antibody and one or more peptides in the third population,        wherein formation of both the first and third sets of        antibody-peptide complexes indicates that the subject is        infected with E. canis and/or E. chaffeensis, and wherein        formation of the first but not the third set of antibody-peptide        complexes indicates that the subject is infected with E.        ewingii.

In yet other embodiments, the method for identifying the species ofEhrlichia infecting a subject, if present, comprises:

-   -   contacting a sample from the subject with a first population of        isolated peptides as described herein;    -   detecting formation of a first set of complexes comprising an        antibody and one or more peptides in the first population;    -   contacting said sample with a second population of isolated        peptides as described herein;    -   detecting formation of a second set of complexes comprising an        antibody and one or more peptides in the second population;    -   contacting said sample with a third population of isolated        peptides as described herein; and    -   detecting formation of a third set of complexes comprising an        antibody and one or more peptides in the third population,        wherein formation of both the first and second sets of complexes        but not the third set indicates that the subject is infected        with E. ewingii, and wherein formation of both the first and        third sets of complexes but not the second set indicates that        the subject is infected with E. canis and/or E. chaffeensis.

In particular embodiments of the methods of the invention, the firstpopulation of isolated peptides is capable of specifically binding toantibodies against antigens from multiple species of Ehrlichia,including E. canis, E. chaffeensis, and E. ewingii. In certainembodiments, the first population of isolated peptides comprises atleast three different peptides, each comprising a sequenceS-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-X₃₉-A-E-T-R-X₄₄-T-F-G-L-X₄₉-K-Q-Y-D-G-A-X₅₆-I-X₅₈-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 1) or a fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₅ is an amino acidselected from the group consisting of E and D, X₈ is an amino acidselected from the group consisting of T and P, X₁₀ is an amino acidselected from the group consisting of T and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₂ is an amino acidselected from the group consisting of L and V, X₁₃ is an amino acidselected from the group consisting of Y and F, X₁₈ is an amino acidselected from the group consisting of D and N, X₂₀ is an amino acidselected from the group consisting of D and N, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₃ is an amino acidselected from the group consisting of A, S, and T, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S, N, and K, X₃₉ is any aminoacid, X₄₄ is any amino acid, X₄₉ is any amino acid, X₅₆ is any aminoacid, and X₅₈ is any amino acid.

In certain embodiments, X₃₉ in SEQ ID NO: 1 is K. In some embodiments,X₄₄ in SEQ ID NO: 1 is K or R, and/or X₄₉ in SEQ ID NO: 1 is E or D. Incertain embodiments, X₅₆ in SEQ ID NO: 1 is K or Q, and/or X₅₈ in SEQ IDNO: 1 is E or T.

In certain other embodiments, the fragment of SEQ ID NO: 1 comprises atleast 20, 25, 30, 35, or 40 contiguous amino acids from SEQ ID NO: 1. Incertain embodiments, the fragment of SEQ ID NO: 1 comprises amino acids33 to 71 of SEQ ID NO: 1. In particular embodiments, each peptide in thefirst population comprises a sequence of SEQ ID NO: 1.

In some embodiments, the first population of isolated peptides comprisesat least one sequence, or a fragment thereof, selected from the groupconsisting of:

(SEQ ID NO: 4) S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 5)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-R-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 6)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-R-T-F-G-L-D-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 7)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-R-T-F-G-L-E-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 8)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-D-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 9)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-D-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 10)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 11)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-T-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 12)S-V-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-T-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 13)S-A-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-T-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 14)S-V-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 15)S-V-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 16)S-A-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 17)S-A-K-E-E-K-Q-P-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 18)S-A-K-E-E-K-Q-P-T-T-A-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 19)S-A-K-E-E-K-Q-P-T-T-G-V-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 20)S-A-K-E-E-K-Q-T-T-T-A-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 21)S-A-K-E-E-K-Q-T-T-T-A-V-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 22)S-A-K-E-E-K-Q-T-T-T-G-V-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 23)S-A-K-E-E-K-Q-T-T-T-G-L-F-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 24)S-A-K-E-E-K-Q-T-T-T-G-L-F-G-L-K-Q-N-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 25)S-A-K-E-E-K-Q-T-T-T-G-L-F-G-L-K-Q-D-W-N-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 26)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-N-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 27)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-N-W-N-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 28)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-N-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 29)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 30)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-S-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 31)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-T-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 32)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-S-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 33)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-T-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 34)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-S-I-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 35)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-T-I-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 36)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-I-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 37)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 38)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-N-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 39)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-K-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 40)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-N-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 41)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-K-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 42)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-N-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-R-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 43)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-K-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-R-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 44)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-R-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 45)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-Q-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 46)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-Q-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 47)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-N-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 48)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-R-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 49)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-E-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 50)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-D-E-N-Q-V-Q-N-K-F-T-I-S-N-C;  and (SEQ ID NO: 51)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-S-E-N-Q-V-Q-N-K-F-T-I-S-N-C.

In some embodiments, the first population of isolated peptides comprisesat least two or three different sequences, or fragments thereof,selected from the group consisting of SEQ ID NOs: 4-51.

In particular embodiments of the methods, the second population ofisolated peptides is capable of specifically or preferentially bindingto antibodies against antigens from E. ewingii. In some embodiments, thesecond population of isolated peptides does not bind or minimally bindsto antibodies against antigens from E. canis or E. chaffeensis. Incertain embodiments, the second population of isolated peptidescomprises at least three different peptides, each comprising a sequenceofF-S-A-K-E-E-X₇-A-E-T-R-X₁₂-T-F-G-L-X₁₇-K-Q-Y-D-G-A-X₂₄-I-X₂₆-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 2) or a fragment thereof, wherein X₇ is any amino acid, X₁₂is any amino acid, X₁₇ is any amino acid, X₂₄ is any amino acid, and X₂₆is any amino acid.

In certain embodiments of the second population of isolated peptides, X₇in SEQ ID NO: 2 is K. In some embodiments, X₁₂ in SEQ ID NO: 2 is K orR, and/or X₁₇ in SEQ ID NO: 2 is E or D. In certain embodiments, X₂₄ inSEQ ID NO: 2 is K or Q, and/or X₂₆ in SEQ ID NO: 2 is E or T.

In certain other embodiments, the fragment of SEQ ID NO: 2 comprises atleast 15, 20, 25, 30, or 35 contiguous amino acids from SEQ ID NO: 2. Insome embodiments, each peptide in the second population comprises asequence of SEQ ID NO: 2.

In particular embodiments, the second population of isolated peptidescomprises at least one sequence, or a fragment thereof, selected fromthe group consisting of:

(SEQ ID NO: 52) F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 53)F-S-A-K-E-E-K-A-E-T-R-R-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 54)F-S-A-K-E-E-K-A-E-T-R-R-T-F-G-L-E-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 55)F-S-A-K-E-E-K-A-E-T-R-R-T-F-G-L-E-K-Q-Y-D-G-A-K-I-T-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 56)F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 57)F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-Q-I-T-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 58)F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-T-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 59)F-S-A-K-E-E-R-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 60)F-S-A-K-E-E-K-A-E-T-R-Q-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 61)F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-Q-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 62)F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-N-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 63)F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-R-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 64)F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-E-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 65)F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-D-E-N-Q-V-Q-N-K-F-T-I-S-N-C;  and (SEQ ID NO: 66)F-S-A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-S-E-N-Q-V-Q-N-K-F-T-I-S-N-C.

In some embodiments, the second population of isolated peptidescomprises at least two or three different sequences, or fragmentsthereof, selected from the group consisting of SEQ ID NOs: 52-66.

In yet other embodiments of the method, the third population of isolatedpeptides is capable of specifically or preferentially binding toantibodies against antigens from E. canis and E. chaffeensis. In someembodiments, the third population of isolated peptides does not bind orminimally binds to antibodies against antigens from E. ewingii. In someembodiments, the third population of isolated peptides comprises atleast three different peptides, each comprising a sequence ofS-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-X₃₉-A-X₄₁-T-R-X₄₄-T-F-G-X₄₈-X₄₉-K-Q-Y-D-G-A-X₅₆-I-X₅₈-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 3) or a fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₅ is an amino acidselected from the group consisting of E and D, X₈ is an amino acidselected from the group consisting of T and P, X₁₀ is an amino acidselected from the group consisting of T and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₂ is an amino acidselected from the group consisting of L and V, X₁₃ is an amino acidselected from the group consisting of Y and F, X₁₈ is an amino acidselected from the group consisting of D and N, X₂₀ is an amino acidselected from the group consisting of D and N, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₃ is an amino acidselected from the group consisting of A, S, and T, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S, N, and K, X₃₉ is any aminoacid, X₄₁ is an amino acid selected from the group consisting of D andN, X₄₄ is any amino acid, X₄₈ is an amino acid selected from the groupconsisting of V and A, X₄₉ is any amino acid, X₅₆ is any amino acid, andX₅₈ is any amino acid.

In certain embodiments of the third population of isolated peptides, X₃₉in SEQ ID NO: 3 is K. In certain embodiments, X₄₄ in SEQ ID NO: 3 is Kor R, and/or X₄₉ in SEQ ID NO: 3 is E or D. In certain embodiments, X₅₆in SEQ ID NO: 3 is K or Q, and/or X₅₈ in SEQ ID NO: 3 is E or T.

In certain other embodiments, the fragment of SEQ ID NO: 3 comprises atleast 20, 25, 30, 35, or 40 contiguous amino acids from SEQ ID NO: 3. Incertain embodiments, the fragment of SEQ ID NO: 3 comprises amino acids33 to 71 of SEQ ID NO: 3. In particular embodiments, each peptide in thethird population comprises a sequence of SEQ ID NO: 3.

In particular embodiments, the third population of isolated peptidescomprises at least one sequence, or a fragment thereof, selected fromthe group consisting of:

(SEQ ID NO: 67) S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 68)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-R-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 69)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-R-T-F-G-V-D-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 70)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-R-T-F-G-V-E-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 71)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-D-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 72)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-D-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 73)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 74)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-T-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 75)S-V-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-T-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 76)S-A-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-T-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 77)S-V-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 78)S-V-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 79)S-A-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 80)S-A-K-E-E-K-Q-P-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 81)S-A-K-E-E-K-Q-P-T-V-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 82)S-A-K-E-E-K-Q-P-T-T-A-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 83)S-A-K-E-E-K-Q-T-T-V-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 84)S-A-K-E-E-K-Q-T-T-V-A-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 85)S-A-K-E-E-K-Q-T-T-T-A-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 86)S-A-K-E-E-K-Q-T-T-T-G-V-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 87)S-A-K-E-E-K-Q-T-T-T-G-V-F-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 88)S-A-K-E-E-K-Q-T-T-T-G-V-Y-G-L-K-Q-N-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 89)S-A-K-E-E-K-Q-T-T-T-G-L-F-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 90)S-A-K-E-E-K-Q-T-T-T-G-L-F-G-L-K-Q-N-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 91)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-N-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 92)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-N-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 93)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-N-G-S-S-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 94)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-N-G-S-T-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 95)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 96)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-S-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 97)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-T-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 98)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-S-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 99)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-T-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 100)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-I-T-N-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 101)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-I-T-K-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 102)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-I-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 103)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 104)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-N-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 105)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-K-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 106)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-N-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 107)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-K-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 108)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-N-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 109)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-N-T-R-K-T-F-G-A-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 110)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-N-T-R-K-T-F-G-V-D-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 111)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-A-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 112)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-A-D-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 113)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-R-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 114)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-Q-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 115)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-Q-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 116)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-N-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 117)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-R-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 118)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-E-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C; (SEQ ID NO: 119)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-D-E-N-Q-V-Q-N-K-F-T-I-S-N-C;  and (SEQ ID NO: 120)S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S-A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-S-E-N-Q-V-Q-N-K-F-T-I-S-N-C.

In some embodiments, the third population of isolated peptides comprisesat least two or three different sequences, or fragments thereof,selected from the group consisting of SEQ ID NOs: 67-120.

In certain embodiments, the populations of isolated peptides used in themethod comprise a fragment of a peptide sequence described herein. Forexample, in certain embodiments, the populations of isolated peptidescomprise a fragment of a sequence selected from the group consisting ofSEQ ID NOs: 1-120. The fragment can be, e.g., at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44amino acids in length. The fragment can be contiguous or can include oneor more deletions (e.g., a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore amino acid residues). In some embodiments, the fragments compriseamino acids 1 to 26 of a sequence selected from the group consisting ofSEQ ID NOs: 1-120. In other embodiments, the fragments comprise aminoacids 33 to 71 of a sequence selected from the group consisting of SEQID NOs: 1, 3, 4-51, and 67-120. In certain embodiments, the fragmentscomprise an epitope of a peptide sequence selected from the groupconsisting of SEQ ID NOs: 1-120.

In some embodiments, one or more of the peptides in the first and/orthird population of peptides used in the method are no longer than 71,75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800,900, 1000, 1200, 1500, or 2000 amino acids in length. In particularembodiments, at least three peptides in the first and/or thirdpopulation of peptides are no longer than 71, 75, 80, 85, 90, 95, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or2000 amino acids in length. In certain embodiments, each peptide in thefirst and/or third population of peptides is no longer than 71, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000,1200, 1500, or 2000 amino acids in length.

In some other embodiments, one or more of the peptides in the secondpopulation of peptides used in the method are no longer than 39, 40, 45,50, 55, 60, 65, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700,800, 900, 1000, 1200, 1500, or 2000 amino acids in length. In particularembodiments, at least three peptides in the second population ofpeptides are no longer than 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or2000 amino acids in length. In certain embodiments, each peptide in thesecond population of peptides is no longer than 39, 40, 45, 50, 55, 60,65, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900,1000, 1200, 1500, or 2000 amino acids in length.

In particular embodiments, each peptide in the first and thirdpopulation of peptides is no longer than 71, 75, 80, 85, 90, 95, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or2000 amino acids in length, and each peptide in the second population ofpeptides is no longer than 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or2000 amino acids in length.

In yet other embodiments, the populations of isolated peptides cancomprise the peptides disclosed in U.S. application Ser. No. 14/052,296and/or US Patent Application Publication No. 2011/0124125A1, thecontents of which are hereby incorporated by reference in theirentirety.

In some embodiments, the peptides in the populations of isolatedpeptides used in the method can comprise a sequence that is at leastabout 80, 85, 90, 95, 98, or 99% identical to a sequence selected fromSEQ ID NOs: 1-120. Percent sequence identity has an art recognizedmeaning and there are a number of methods to measure identity betweentwo polypeptide or polynucleotide sequences. See, e.g., Lesk, Ed.,Computational Molecular Biology, Oxford University Press, New York,(1988); Smith, Ed., Biocomputing: Informatics And Genome Projects,Academic Press, New York, (1993); Griffin & Griffin, Eds., ComputerAnalysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); vonHeinje, Sequence Analysis In Molecular Biology, Academic Press, (1987);and Gribskov & Devereux, Eds., Sequence Analysis Primer, M StocktonPress, New York, (1991). Methods for aligning polynucleotides orpolypeptides are codified in computer programs, including the GCGprogram package (Devereux et al., Nuc. Acids Res. 12:387 (1984)),BLASTP, BLASTN, FASTA (Atschul et al., J Molec. Biol. 215:403 (1990)),and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711) which uses the local homology algorithm ofSmith and Waterman (Adv. App. Math., 2:482-489 (1981)). For example, thecomputer program ALIGN which employs the FASTA algorithm can be used,with an affine gap search with a gap open penalty of −12 and a gapextension penalty of −2.

When using any of the sequence alignment programs to determine whether aparticular sequence is, for instance, about 95% identical to a referencesequence, the parameters are set such that the percentage of identity iscalculated over the full length of the reference polypeptide and thatgaps in identity of up to 5% of the total number of amino acids in thereference polypeptide are allowed.

Variants of the peptide sequences can be readily selected by one ofskill in the art, based in part on known properties of the sequence. Forexample, a variant peptide can include amino acid substitutions (e.g.,conservative substitutions with naturally occurring amino acids,non-naturally occurring amino acids, or amino acid analogs) and/ordeletions (e.g., small, single amino acid deletions, or deletionsencompassing 2, 3, 4, 5, 10, 15, 20, or more contiguous amino acids).Thus, in certain embodiments, a variant of a native peptide sequence isone that differs from a naturally-occurring sequence by (i) one or more(e.g., 2, 3, 4, 5, 6, or more) conservative amino acid substitutions,(ii) deletion of 1 or more (e.g., 2, 3, 4, 5, 6, or more) amino acids,or (iii) a combination thereof. Deleted amino acids can be contiguous ornon-contiguous. Conservative amino acid substitutions are those thattake place within a family of amino acids that are related in their sidechains and chemical properties. These include, e.g., (1) acidic aminoacids: aspartate, glutamate; (2) basic amino acids: lysine, arginine,histidine; (3) nonpolar amino acids: alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan; (4)uncharged polar amino acids: glycine, asparagine, glutamine, cysteine,serine, threonine, tyrosine; (5) aliphatic amino acids: glycine,alanine, valine, leucine, isoleucine, serine, threonine, with serine andthreonine optionally grouped separately as aliphatic-hydroxyl; (6)aromatic amino acids: phenylalanine, tyrosine, tryptophan; (7) amideamino acids: asparagine, glutamine; and (9) sulfur-containing aminoacids: cysteine and methionine. See, e.g., Biochemistry, 2nd ed., Ed. byL. Stryer, W H Freeman and Co: 1981. Methods for confirming that variantpeptides are suitable are conventional and routine.

Variants of the peptide sequences encompass variations on previouslydefined peptide sequences. For example, a previously described peptidesequence comprising a known epitope may be lengthened or shortened, atone or both ends (e.g., by about 1-3 amino acids), and/or one, two,three, four or more amino acids may be substituted by conservative aminoacids, etc. Furthermore, if a region of a protein has been identified ascontaining an epitope of interest, an investigator can “shift” theregion of interest (e.g., by about 5 amino acids in either direction)from the endpoints of the original rough region to optimize theactivity.

In some embodiments, the peptides in the populations of isolatedpeptides used in the method can further comprise an additionalN-terminal peptide sequence, an additional C-terminal peptide sequence,or a combination thereof.

In certain embodiments, the additional N-terminal peptide sequence cancomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids and can beeither a native or non-native sequence. In other embodiments, theadditional C-terminal peptide sequence can comprise 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more amino acids and can be either a native or non-nativesequence.

The additional N-terminal or C-terminal peptide sequence can be a nativesequence. As used herein, a “native” sequence is a peptide sequence froma naturally-occurring Ehrlichia OMP-1 sequence, or a variant thereof. Incertain embodiments, the peptide sequence is a fragment of anaturally-occurring Ehrlichia OMP-1 sequence. The peptide sequence canbe, e.g., from a conserved or non-conserved region of OMP-1. The peptidesequence can comprise, e.g., an epitope, such as an immunodominantepitope or any other epitope recognizable by a host (e.g., human, dog,etc.) immune system. OMP-1 proteins and peptides thereof have beendescribed, e.g., in U.S. Pat. Nos. 6,544,517, 6,893,640, 6,923,963,7,063,846, and 7,407,770, U.S. Patent Applications 2004/0265333 and2009/0075368, and European Patent No. 1026949, the contents of each ofwhich are incorporated herein by reference in their entirety.

In certain embodiments, the additional N-terminal or C-terminal peptidesequence is a non-native sequence. As used herein, a “non-native”sequence is any protein sequence, whether from an Ehrlichia protein orotherwise, other than a native OMP-1 peptide sequence.

In certain embodiments, the additional N-terminal or C-terminal peptidesequence can comprise or consist of another peptide having a sequence,or a fragment thereof, selected from SEQ ID NOs: 1-120.

In some embodiments, the additional N-terminal or C-terminal peptidesequence can be linked to the peptides in the populations of isolatedpeptides through one or more linking amino acids (e.g. glycine, serine,or cysteine residues).

The isolated peptides in the populations may be isolated by chemicalsynthesis and/or purification. In some embodiments, the peptides areproduced biologically (i.e., by cellular machinery, such as a ribosome)and then isolated. As used herein, an “isolated” peptide is a peptidethat has been produced either synthetically or biologically and thenpurified, at least partially, from the chemicals and/or cellularmachinery used to produce the peptide. In certain embodiments, anisolated peptide of the invention is substantially purified. The term“substantially purified,” as used herein, refers to a molecule, such asa peptide, that is substantially free of cellular material (proteins,lipids, carbohydrates, nucleic acids, etc.), culture medium, chemicalprecursors, chemicals used in synthesis of the peptide, or combinationsthereof. A peptide that is substantially purified has less than about40%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less of the cellularmaterial, culture medium, other polypeptides, chemical precursors,and/or chemicals used in synthesis of the peptide. Accordingly, asubstantially pure molecule, such as a peptide, can be at least about60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, by dry weight, themolecule of interest. An isolated peptide or population of peptides canbe in water, a buffer, or in a dry form awaiting reconstitution, e.g.,as part of a kit.

In certain embodiments, one or more peptides in the populations areconjugated to a ligand. For example, in certain embodiments, thepeptides are biotinylated. In other embodiments, the peptides areconjugated to streptavidin, avidin, or neutravidin. In otherembodiments, the peptides are conjugated to a carrier protein (e.g.,serum albumin, keyhole limpet hemocyanin (KLH), or an immunoglobulin Fcdomain). In still other embodiments, the peptides are conjugated to adendrimer and/or are part of a multiple antigenic peptide system (MAPS).The peptides may also be conjugated to colloidal gold, quantum dots orother nanoparticles and/or to latex particles. In still anotherembodiment, the peptides may be conjugated to enzymes, fluorescent orchemi-luminescent markers.

In certain embodiments, peptides in the populations of isolated peptidesare modified. The peptides of the invention may be modified by a varietyof techniques, such as by denaturation with heat and/or a detergent(e.g., SDS). Alternatively, peptides of the invention may be modified byassociation with one or more further moieties. The association can becovalent or non-covalent, and can be, for example, via a terminal aminoacid linker, such as lysine or cysteine, a chemical coupling agent, or apeptide bond. The additional moiety can be, for example, a ligand, aligand receptor, a fusion partner, a detectable label, an enzyme, or asubstrate that immobilizes the peptide.

In addition, the peptides in the populations of isolated peptides may bemodified to include any of a variety of known chemical groups ormolecules. Such modifications include, but are not limited to,glycosylation, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment to polyethylene glycol (e.g., PEGylation), covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation, gammacarboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, ubiquitination, modifications with fatty acids, transfer-RNAmediated addition of amino acids to proteins such as arginylation, etc.Analogues of an amino acid (including unnatural amino acids) andpeptides with substituted linkages are also included.

Modifications as set forth above are well-known to those of skill in theart and have been described in great detail in the scientificliterature. Several particularly common modifications, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, for instance, aredescribed in many basic texts, such as Proteins-Structure and MolecularProperties, 2nd ed., T. e. Creighton, W.H. Freeman and Company, New York(1993). Many detailed reviews are available on this subject, such as byWold, F., Posttranslational Covalent Modification of Proteins, B. C.Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.(1990) Meth. Enzymol. 182:626-646 and Rattan et al. (1992) Ann. N.Y.Acad. Sci. 663:48-62.

In certain embodiments, one or more or all peptides in a population ofpeptides is attached to or immobilized on a substrate, such as a solidor semi-solid support. The attachment can be covalent or non-covalent,and can be facilitated by a moiety associated with the peptide thatenables covalent or non-covalent binding, such as a moiety that has ahigh affinity to a component attached to the carrier, support orsurface. For example, the peptide can be associated with a ligand, suchas biotin, and the component associated with the surface can be acorresponding ligand receptor, such as avidin. In some embodiments, thepeptide can be associated with a fusion partner, e.g., bovine serumalbumin (BSA), which facilitates the attachment of the peptide to asubstrate. In other embodiments, the peptides of the invention areattached to or immobilized on a substrate via a metallic nanolayer suchas a localized surface plasmon resonance spectroscopy (LSPR) surface. Inone embodiment, the metallic nanolayer is comprised of cadmium, zinc,mercury, or a noble metal, such as gold, silver, copper, and platinum.The peptide or population of peptides can be attached to or immobilizedon the substrate either prior to or after the addition of a samplecontaining antibody during an immunoassay.

In certain embodiments, the substrate is a bead, such as a colloidalparticle (e.g., a colloidal nanoparticle made from gold, silver,platinum, copper, cadmium, metal composites, other soft metals,core-shell structure particles, or hollow gold nanospheres) or othertype of particle (e.g., a magnetic bead or a particle or nanoparticlecomprising silica, latex, polystyrene, polycarbonate, polyacrylate, orPVDF). Such particles can comprise a label (e.g., a colorimetric,chemiluminescent, or fluorescent label) and can be useful forvisualizing the location of the peptides during immunoassays. In certainembodiments, a terminal cysteine of a peptide of the invention is usedto bind the peptide directly to the nanoparticles made from gold,silver, platinum, copper, cadmium, metal composites, or other softmetals, or metallic nanoshells (e.g., gold hollow spheres, gold-coatedsilica nanoshells, and silica-coated gold shells).

In certain embodiments, the substrate is a dot blot or a flow path in alateral flow immunoassay device. For example, the peptides can beattached or immobilized on a porous membrane, such as a PVDF membrane(e.g., an Immobilon™ membrane), a nitrocellulose membrane, polyethylenemembrane, nylon membrane, or a similar type of membrane.

In certain embodiments, the substrate is a flow path in an analytical orcentrifugal rotor. In other embodiments, the substrate is a tube or awell, such as a well in a plate (e.g., a microtiter plate) suitable foruse in an ELISA assay. Such substrates can comprise glass,cellulose-based materials, thermoplastic polymers, such as polyethylene,polypropylene, or polyester, sintered structures composed of particulatematerials (e.g., glass or various thermoplastic polymers), or castmembrane film composed of nitrocellulose, nylon, polysulfone, or thelike. A substrate can be sintered, fine particles of polyethylene,commonly known as porous polyethylene, for example, 0.2-15 micron porouspolyethylene from Chromex Corporation (Albuquerque, N. Mex.). All ofthese substrate materials can be used in suitable shapes, such as films,sheets, or plates, or they may be coated onto or bonded or laminated toappropriate inert carriers, such as paper, glass, plastic films, orfabrics. Suitable methods for immobilizing peptides on solid phasesinclude ionic, hydrophobic, covalent interactions and the like.

In one embodiment, the methods of invention involve detecting thepresence of naturally occurring antibodies against one or more Ehrlichiaantigens (e.g., the antigen of a pathogenic Ehrlichia, such as E.chaffeensis, E. muris, E. ewingii, or E. canis) which are produced bythe infected subject's immune system in its biological fluids ortissues, and which are capable of binding specifically to one of morepeptides in a population of peptides and, optionally, one or moresuitable additional antigenic polypeptides or peptides.

For example, in one aspect, the present invention provides a method ofdetecting in a sample from a subject the presence of antibodies againstantigens from E. chaffeensis, E. muris, E. ewingii, and/or E. caniscomprising contacting the sample with a population of peptidescomprising at least three different peptides, wherein each peptidecomprises a sequence of SEQ ID NO: 1; and detecting formation ofcomplexes comprising an antibody and one or more peptides in thepopulation, wherein formation of the complexes indicates the presence ofantibodies against antigens from E. chaffeensis, E. muris, E. ewingii,and/or E. canis. In some embodiments, the population of peptidescomprises at least two or three different sequences selected from thegroup consisting of SEQ ID NOs: 4-51.

In other embodiments, the present invention provides a method ofdetecting in a sample from a subject the presence of antibodies againstantigens from E. ewingii comprising contacting the sample with apopulation of peptides comprising at least three different peptides,wherein each peptide comprises a sequence of SEQ ID NO: 2; and detectingformation of complexes comprising an antibody and one or more peptidesin the population, wherein formation of the complexes indicates thepresence of antibodies against antigens from E. ewingii. In someembodiments, the population of peptides comprises at least two or threedifferent sequences selected from the group consisting of SEQ ID NOs:52-66.

In certain embodiments, the present invention provides a method ofdetecting in a sample from a subject the presence of antibodies againstantigens from E. chaffeensis and/or E. canis comprising contacting thesample with a population of peptides comprising at least three differentpeptides, wherein each peptide comprises a sequence of SEQ ID NO: 3; anddetecting formation of complexes comprising an antibody and one or morepeptides in the population, wherein formation of the complexes indicatesthe presence of antibodies against antigens from E. chaffeensis and/orE. canis. In some embodiments, the population of peptides comprises atleast two or three different sequences selected from the groupconsisting of SEQ ID NOs: 67-120.

There are a number of different assays that may be used to detectformation of antibody-peptide complexes comprising one or more peptidesin the methods of the invention. For example, the detecting step cancomprise performing an ELISA assay, performing an immunofluorescenceassay, performing a lateral flow immunoassay, performing anagglutination assay, performing a wavelength shift assay, performing aWestern blot, slot blot, or dot blot, analyzing the sample in ananalytical or centrifugal rotor, or analyzing the sample with anelectrochemical, optical, or opto-electronic sensor. These differentassays are described herein and/or are well-known to those skilled inthe art.

Suitable immunoassay methods typically include: receiving or obtaining(e.g., from a patient) a sample of body fluid or tissue likely tocontain antibodies; contacting (e.g., incubating or reacting) a sampleto be assayed with a population of peptides, under conditions effectivefor the formation of a specific peptide-antibody complex (e.g., forspecific binding of the peptide to the antibody); and assaying thecontacted (reacted) sample for the presence of an antibody-peptidereaction (e.g., determining the amount of an antibody-peptide complex).The presence of an elevated amount of the antibody-peptide complexindicates that the subject was exposed to and infected with aninfectious Ehrlichia species. A peptide, including a modified formthereof, which “binds specifically” to (e.g., “is specific for” or binds“preferentially” to) an antibody against an Ehrlichia antigen interactswith the antibody, or forms or undergoes a physical association with it,in an amount and for a sufficient time to allow detection of theantibody. By “specifically” or “preferentially,” it is meant that thepeptide has a higher affinity (e.g., a higher degree of selectivity) forsuch an antibody than for other antibodies in a sample. For example, thepeptide can have an affinity for the antibody of at least about1.5-fold, 2-fold, 2.5-fold, 3-fold, or higher than for other antibodiesin the sample. Such affinity or degree of specificity can be determinedby a variety of routine procedures, including, e.g., competitive bindingstudies. In an ELISA assay, a positive response is defined as a value 2or 3 standard deviations greater than the mean value of a group ofhealthy controls. In some embodiments, a second tier assay is requiredto provide an unequivocal serodiagnosis of monocytic and/or granulocyticehrlichiosis.

Phrases such as “sample containing an antibody” or “detecting anantibody in a sample” are not meant to exclude samples or determinations(e.g., detection attempts) where no antibody is contained or detected.In a general sense, this invention involves assays to determine whetheran antibody produced in response to infection with an infectiousEhrlichia is present in a sample, irrespective of whether or not it isdetected.

Conditions for reacting peptides and antibodies so that they reactspecifically are well-known to those of skill in the art. See, e.g.,Current Protocols in Immunology (Coligan et al., editors, John Wiley &Sons, Inc).

In some embodiments, the methods comprise receiving or obtaining asample of body fluid or tissue likely to contain antibodies from asubject. The antibodies can be, e.g., of IgG, IgE, IgD, IgM, or IgAtype. Generally, IgM and/or IgA antibodies are detected, e.g., fordetection at early stages of infection. IgG antibodies can be detectedwhen some of the additional peptides discussed above are used in themethod (e.g., peptides for the detection of flagellum proteins). Thesample is preferably easy to obtain and may be whole blood, plasma, orserum derived from a venous blood sample or even from a finger prick.Tissue from other body parts or other bodily fluids, such ascerebro-spinal fluid (CSF), saliva, gastric secretions, mucus, urine,etc., are known to contain antibodies and may be used as a source of thesample. The sample may also be a tissue extract or a cell lysate.

Once a population of peptides and sample antibody are permitted to reactin a suitable medium, an assay is performed to determine the presence orabsence of an antibody-peptide reaction. Among the many types ofsuitable assays, which will be evident to a skilled worker, areimmunoprecipitation and agglutination assays performed with or withoutenhancement.

The protocols for immunoassays using antigens for detection of specificantibodies are well known in art. For example, a conventional sandwichassay can be used, or a conventional competitive assay format can beused. For a discussion of some suitable types of assays, see CurrentProtocols in Immunology (supra). In certain embodiments, a peptide ofthe invention is immobilized on a solid or semi-solid surface or carrierby means of covalent or non-covalent binding, either prior to or afterthe addition of the sample containing antibody.

Devices for performing specific binding assays, especially immunoassays,are known and can be readily adapted for use in the present methods.Solid phase assays, in general, are easier to perform than heterogeneousassay methods which require a separation step, such as precipitation,centrifugation, filtration, chromatography, or magnetism, becauseseparation of reagents is faster and simpler. Solid-phase assay devicesinclude microtiter plates, flow-through assay devices (e.g., lateralflow immunoassay devices), dipsticks, and immunocapillary orimmunochromatographic immunoassay devices.

In some embodiments of the invention, the solid or semi-solid surface orcarrier attached to the populations of peptides is the floor or wall ina microtiter well, a filter surface or membrane (e.g., a nitrocellulosemembrane or a PVDF (polyvinylidene fluoride) membrane, such as anImmobilon™ membrane), a hollow fiber, a beaded chromatographic medium(e.g., an agarose or polyacrylamide gel), a magnetic bead, a fibrouscellulose matrix, an HPLC matrix, an FPLC matrix, a substance havingmolecules of such a size that the molecules with the peptide boundthereto, when dissolved or dispersed in a liquid phase, can be retainedby means of a filter, a substance capable of forming micelles orparticipating in the formation of micelles allowing a liquid phase to bechanged or exchanged without entraining the micelles, a water-solublepolymer, or any other suitable carrier, support or surface.

In some embodiments of the invention, a population of peptides isprovided with a suitable label which enables detection. Conventionallabels may be used which are capable, alone or in concert with othercompositions or compounds, of providing a detectable signal. Suitablelabels include, but are not limited to, enzymes (e.g., HRP,beta-galactosidase, alkaline phosphatase, etc.), fluorescent labels,radioactive labels, colored latex particles, and metal-conjugated labels(e.g., metallic nanolayers, metallic nanoparticle- or metallicnanoshell-conjugated labels). Suitable metallic nanoparticle or metallicnanoshell labels include, but are not limited to, gold particles, silverparticles, copper particles, platinum particles, cadmium particles,composite particles, gold hollow spheres, gold-coated silica nanoshells,and silica-coated gold shells. Metallic nanolayers suitable fordetectable layers include nanolayers comprised of cadmium, zinc,mercury, and noble metals, such as gold, silver, copper, and platinum.

Suitable detection methods include, e.g., detection of an agent which istagged, directly or indirectly, with a colorimetric assay (e.g., fordetection of HRP or beta-galactosidase activity), visual inspectionusing light microscopy, immunofluorescence microscopy, includingconfocal microscopy, or by flow cytometry (FACS), autoradiography (e.g.,for detection of a radioactively labeled agent), electron microscopy,immunostaining, subcellular fractionation, or the like. In oneembodiment, a radioactive element (e.g., a radioactive amino acid) isincorporated directly into a peptide chain; in another embodiment, afluorescent label is associated with a peptide via biotin/avidininteraction, association with a fluorescein conjugated antibody, or thelike. In one embodiment, a detectable specific binding partner for theantibody is added to the mixture. For example, the binding partner canbe a detectable secondary antibody or other binding agent (e.g., proteinA, protein G, protein L, chimeric proteins A/G, A/G/L, A/L, G/L orcombinations thereof) which binds to the first antibody. This secondaryantibody or other binding agent can be labeled, e.g., with aradioactive, enzymatic, fluorescent, luminescent, chemi-luminescent,metallic nanoparticle or metallic nanoshell (e.g. colloidal gold), orother detectable label, such as an avidin/biotin, avidin/streptavidin oravidin/polystreptavidin system. In another embodiment, the bindingpartner is a peptide of the invention, which can be conjugated directlyor indirectly (e.g. via biotin/avidin or biotin/streptavidininteraction) to an enzyme, such as horseradish peroxidase or alkalinephosphatase or other signaling moiety. In such embodiments, thedetectable signal is produced by adding a substrate of the enzyme thatproduces a detectable signal, such as a chromogenic, fluorogenic, orchemiluminescent substrate.

A “detection system” for detecting bound peptide, as used herein, maycomprise a detectable binding partner, such as an antibody specific forthe peptide. In one embodiment, the binding partner is labeled directly.In another embodiment, the binding partner is attached to a signalgenerating reagent, such as an enzyme that, in the presence of asuitable substrate, can produce a detectable signal. A surface forimmobilizing the peptide may optionally accompany the detection system.

In some embodiments of the invention, the detection procedure comprisesvisibly inspecting the antibody-peptide complex for a color change, orinspecting the antibody-peptide complex for a physical-chemical change.Physical-chemical changes may occur with oxidation reactions or otherchemical reactions. They may be detected by eye, using aspectrophotometer, or the like.

A very useful assay format is a lateral flow immunoassay format.Antibodies to human or animal (e.g., dog, mouse, deer, etc.)immunoglobulins, or staph A, G, or L proteins, can be labeled with asignal generator or reporter (e.g., colloidal gold) that is dried andplaced on a glass fiber pad (sample application pad or conjugate pad).The diagnostic peptide is immobilized on membrane, such asnitrocellulose or a PVDF (polyvinylidene fluoride) membrane (e.g., anImmobilon™ membrane). When a solution of sample (blood, serum, etc.) isapplied to the sample application pad (or flows through the conjugatepad), it dissolves the labeled reporter, which then binds to allantibodies in the sample. The resulting complexes are then transportedinto the next membrane (PVDF or nitrocellulose containing the diagnosticpeptide) by capillary action. If antibodies against the diagnosticpeptide are present, they bind to the diagnostic peptide striped on themembrane, thereby generating a signal (e.g., a band that can be seen orvisualized). An additional antibody specific to the labeled antibody ora second labeled antibody can be used to produce a control signal.

An alternative format for the lateral flow immunoassay comprises thepopulations of isolated peptides being conjugated to a ligand (e.g.,biotin) and complexed with labeled ligand receptor (e.g.,streptavidin-colloidal gold). The labeled peptide complexes can beplaced on the sample application pad or conjugate pad. Anti-humanIgG/IgM or anti-animal (e.g., dog, mouse, deer) IgG/IgM antibodies orother peptides of the invention are immobilized on a membrane, such asnitrocellulose of PVDF, at a test site (e.g., a test line). When asample is added to the sample application pad, antibodies in the samplereact with the labeled peptide complexes such that antibodies that bindto peptides of the invention become indirectly labeled. The antibodiesin the sample are then transported into the next membrane (PVDF ornitrocellulose containing the diagnostic peptide) by capillary actionand bind to the immobilized anti-human IgG/IgM or anti-animal IgG/IgMantibodies (or protein A, protein G, protein A/G fusion proteins,protein L, or combinations thereof) or immobilized peptides of theinvention. If any of the sample antibodies are bound to the labeledpeptides of the invention, the label associated with the peptides can beseen or visualized at the test site. In another embodiment of this typeof lateral flow device (in which the peptides of the invention are usedboth as the immobilized capture agent at a test site and as a solublelabeled complex to react with antibodies in a sample), to amplify thedetection signal, protein A, protein G, and/or protein A/G fusionproteins conjugated to a detectable label (e.g., metallic nanoparticleor nanoshell, HRP, ALP, fluorophore, colored latex particle) may beapplied to the test site where they will bind to the Fc region of anyantibodies to Ehrlichia antigens captured by the immobilized peptides ofthe invention. Suitable controls for this assay can include, e.g., achicken IgY-colloidal gold conjugate located at the sample applicationpad or conjugate pad, and an anti-chicken IgY antibody immobilized at acontrol site located proximal to the test site. Other suitable controlscan include chicken anti-Protein A, mouse IgG or any other proteinscapable of binding to Protein A/G/L. In at least some of the lateralflow immunoassays performed in the methods of invention and describedherein, chicken anti-Protein A was used as the control line.

Another assay for the screening of blood products or other physiologicalor biological fluids is an enzyme linked immunosorbent assay, i.e., anELISA. Typically in an ELISA, isolated peptides or mixtures orpopulations of peptides are adsorbed directly, or following conjugationto a carrier protein, to the surface of a microtiter well directly orthrough a capture matrix (e.g., an antibody). Residual, non-specificprotein-binding sites on the surface are then blocked with anappropriate agent, such as bovine serum albumin (BSA), heat-inactivatednormal goat serum (NGS), or BLOTTO (a buffered solution of nonfat drymilk which also contains a preservative, salts, and an antifoamingagent). The well is then incubated with a biological sample suspected ofcontaining specific anti-Ehrlichia (e.g., anti-E. chaffeensis, anti-E.ewingii, or anti-E. canis) antibody. Such biological sample can be aserum, plasma, or other type of sample. The sample can be applied neat,or more often it can be diluted, usually in a buffered solution whichcontains a small amount (0.1-10.0% by weight) of protein, such as BSA,NGS, or BLOTTO. After incubating for a sufficient length of time toallow specific binding to occur, the well is washed to remove unboundprotein and then incubated with an optimal concentration of anappropriate anti-immunoglobulin antibody (e.g., for human subjects, ananti-human immunoglobulin (αHuIg) from another animal, such as dog,mouse, cow, etc.) or another peptide of the invention that is conjugatedto an enzyme or other label by standard procedures and is dissolved inblocking buffer. The label can be chosen from a variety of enzymes,including horseradish peroxidase (HRP), beta-galactosidase, alkalinephosphatase (ALP), glucose oxidase, etc. In certain embodiments, ProteinA or Protein G-HRP is used in the methods of invention. Sufficient timeis allowed for specific binding to occur again, then the well is washedagain to remove unbound conjugate, and a suitable substrate for theenzyme is added. Color is allowed to develop and the optical density ofthe contents of the well is determined visually or instrumentally(measured at an appropriate wave length). The cutoff OD value may bedefined as the mean OD+3 standard deviations (SDs) of at least 50 serumsamples collected from individuals from an area where ehrlichiosis isnot endemic, or by other such conventional definitions. In the case of avery specific assay, OD+2 SD can be used as a cutoff value.

In another embodiment, the methods comprise an agglutination assay. Forexample, in certain embodiments, metallic nanoparticles or metallicnanoshells (e.g., colloidal gold, etc.) or latex beads are conjugated tothe populations of isolated peptides. Subsequently, the biological fluidis incubated with the bead/peptide conjugate, thereby forming a reactionmixture. The reaction mixture is then analyzed to determine the presenceof the antibodies. In certain embodiments, the agglutination assayscomprise the use of a second population of particles, such as metallicnanoparticles or metallic nanoshells (e.g., colloidal gold, etc.) orlatex beads, conjugated to (1) antibodies specific to the peptides ofcompositions of the invention, in the case of a competition assay, or(2) antibodies capable of detecting sample antibodies (e.g., anti-humanIgG or IgM antibodies, anti-dog IgG or IgM antibodies, anti-cat IgG orIgM antibodies, etc.), in the case of a sandwich assay. Suitableagglutination methods can comprise centrifugation as a means ofassessing the extent of agglutination.

In still other embodiments, the populations of isolated peptides areelectro- or dot-blotted onto nitrocellulose paper. Subsequently, asample, such as a biological fluid (e.g., serum or plasma) is incubatedwith the blotted antigen, and antibody in the biological fluid isallowed to bind to the antigen(s). The bound antibody can then bedetected, e.g., by standard immunoenzymatic methods or by visualizationusing metallic nanoparticles or nanoshells coupled to secondaryantibodies or other antibody binding agents, such as protein A, proteinG, protein A/G fusion proteins, protein L, or combinations thereof.

In still other embodiments, peptide or compositions of the invention areelectro- or dot-blotted onto nitrocellulose paper. Subsequently, asample, such as a biological fluid (e.g., serum or plasma) is incubatedwith the blotted antigen, and antibody in the biological fluid isallowed to bind to the antigen(s). The bound antibody can then bedetected, e.g., by standard immunoenzymatic methods or by visualizationusing metallic nanoparticles or nanoshells coupled to secondaryantibodies or other antibody binding agents, such as protein A, proteinG, protein A/G fusion proteins, protein L, or combinations thereof.

In still other embodiments, a protein microarray (or protein chip) isused in the methods. For example, in certain embodiments, the microarrayor chip comprises a support surface such as a glass slide,nitrocellulose membrane, bead, or microtitre plate, which are conjugatedto an array of capture proteins comprising a population of peptides asdescribed above. Samples, optionally labeled with a fluorescent dye, areadded to the array. Specific binding between the antibodies in thesamples, if present, and the immobilized protein emits a fluorescentsignal that is read by a laser scanner. Unlabeled antibodies bound tothe peptides of invention may also be subsequently labeled with quantumdot-labeled Protein A, A/G, etc. Microarrays of isolated peptides mayalso be used in a microchip chip format in a centrifugal analyzer.Protein microarrays are high-throughput, rapid, automated, economical,and highly sensitive, consuming small quantities of samples andreagents.

It should be understood by one of skill in the art that any number ofconventional protein assay formats, particularly immunoassay formats,may be designed to utilize the populations of isolated peptides for anyof the methods described herein. This invention is thus not limited bythe selection of the particular assay format, and is believed toencompass all suitable assay formats that are known to those of skill inthe art.

Using any of the suitable assay formats described herein or otherwiseknown to those of skill in the art, formation of complexes comprising anantibody and one or more peptides in the populations of isolatedpeptides can be detected. By a “set” of complexes, it refers tocomplexes formed between one population of isolated peptides and anyantibodies in a sample. When a detection result is described asformation of one but not another set of complexes, it includes a rangeof results that can be obtained with two different populations ofisolated peptides. By “formation of the first but not the second set ofcomplexes”, e.g., it can include a clearly positive result obtained withthe first population of isolated peptides and a clearly negative resultwith the second population of isolated peptides. It can also include avery high score of the result obtained with the first population ofisolated peptides and a very low score of the result obtained with thesecond population of isolated peptides. It can further include anyrelatively higher score of the result obtained with the first populationthan the second population of isolated peptides.

For any of the assay formats described herein, a score can be assignedto the assay result of each sample. Such score refers to a relativevalue, level, strength, or degree of an assay result. It can beartificially created by a person of skill in the art or by using analgorithm, sometimes using samples with known analytes, e.g., antigensor antibodies, optionally using samples with known concentrations ortiters of the known analytes (which can be called “standards” or“calibrators”). A score can be a number manually assigned by a person ofskill in the art or generated with a formula or computer algorithm,e.g., from zero for a negative control to any positive number for apositive control (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 80, 100,120, 150, 200, 300, 400, 500, 1000, etc.). It can also be represented bysymbols, e.g., “−” for a negative control, and “+”, “++”, “+++”, etc.,for positive controls. A score can be determined by calculation with aformula or by automatic processing with a computer algorithm, or can bedetermined by visual inspection, measurement, or estimation of the assayresult. When using samples with known concentrations or titers of knownanalytes (the standards or calibrators), such standards/calibrators canbe assayed in diluted and undiluted conditions, and a range of scores ora standard curve of scores can be generated, which can be used todetermine the scores of unknown samples assayed for the same analytes,preferably with the same assays and in the same assay runs.

In certain embodiments, the method uses a combination of immunochemicalassays and three populations of peptides to identify whether a sample isinfected with one, two or all three of the following Ehrlichia species:E. canis, E. chaffeensis, and E. ewingii.

In some embodiments of the method, a standard sample that has a knowntiter of antibodies against a certain species (e.g., E. ewingii), or acertain combination of species (e.g., E. canis and E. chaffeensis, or E.canis, E. chaffeensis, and E. ewingii) is tested. In certainembodiments, the standard sample is diluted to a series ofstandards/calibrators. Calibrators may be prepared using purifiedantibodies. Calibrators may also be prepared by selecting, poolingand/or diluting antisera/antibody samples with various levels ofantibody titer. A person of skill in the art should know how to generatesuitable calibrators. Scores and a standard curve can be generated forthe series of standards/calibrator. In some embodiments, a cutoff isgenerated for a sample to be classified as positive for comprisingantibodies against certain species of Ehrlichia (e.g., a cutoff forantibodies against E. ewingii, a cutoff for antibodies against E. canisand E. chaffeensis, and another cutoff for antibodies against E. canis,E. chaffeensis, and E. ewingii). A sample can be classified as a low,medium, or high sample. A low sample is usually just above the limit ofdetection, and a very high sample shows most response in a givenpopulation. Calibrators for ELISA are often prepared to represent theranges from low to very high samples.

In certain embodiments, an unknown sample is tested with the same assaysas the standards. In some embodiments, a score of the unknown sample isgenerated against the scores or the standard curve of each standard,e.g., a score of the unknown sample can be generated against a standardthat has a known titer of antibodies against E. ewingii; and anotherscore of the unknown sample can be generated against a standard that hasa known titer of antibodies against both E. canis and E. chaffeensis.

Scores can be compared among samples assayed for a same analyte or fordifferent analytes.

When comparing scores of samples assayed for a same analyte, the scorescan be determined from and compared against the same range of scores orthe same standard curve of scores generated from thestandards/calibrators, if all the samples are assayed in the sameexperiment under the same conditions as the standards/calibrators. Thescores can also be determined from different ranges of scores ordifferent standard curves of scores from the standards/calibrators, ifthe samples are assayed in different experiments along with the samestandards/calibrators. Then relative scores of the assayed samples inrelation to the same standards/calibrators can be determined andcompared to each other.

When comparing scores of samples assayed for different analytes, e.g.,antibodies against different species of Ehrlichia, each species orcombination of species (such as E. canis and E. chaffeensis) has its owncalibrators. The limit of detection for each set of calibrators andassay is determined by generating a cutoff normally assigned by adding2-3 standard deviations to the mean of the samples known to be negativefor the antibodies being detected. The calibrators for the population ofisolated peptides detecting all of the Ehrlichia species contain amixture of antibodies to different Ehrlichia species in appropriateratios, for example, at least 5% each of anti-canis, anti-chaffeensis,and anti-ewingii, so that there is enough of each species and the assaydoes not miss any of the assayed species. The calibrators for thepopulation of peptides detecting both E. canis and E. chaffeensis,contain a mixture of anti-canis and anti-chaffeensis samples inappropriate ratios, for example, at least 5% of each species. Thecalibrators for the population of peptides detecting only E. ewingiicontain only anti-ewingii samples. The calibrators for the population ofpeptides detecting E. canis/E. chaffeensis and for the population ofpeptides detecting E. ewingii are assigned the same range of scoreswhich are limited to the linear portion of the respective standardcurves.

In some embodiments, scores are generated from detecting the formationof complexes comprising antibodies and peptides in the populations ofpeptides as described herein. In certain embodiments, a score isgenerated from detecting the formation of complexes comprisingantibodies in a sample, if present, and peptides in a first, second, orthird population of peptides as described herein, resulting in a firstscore, a second score, or a third score, respectively.

In some embodiments, the second score is compared to the third score,manually or by using a computer. In particular embodiments, a sample isidentified or classified to be infected with E. ewingii if the secondscore is higher than the third score. In other embodiments, a sample isidentified or classified to be infected with E. canis and/or E.chaffeensis if the third score is higher than the second score.

In yet other embodiments, the method further comprises a step todetermine whether the infecting species is E. canis or E. chaffeensis.For example in one such embodiment an assay is performed to detectantibodies against E. canis but not E. chaffeensis, to generate a scorefor E. canis. Another assay can be performed to detect antibodiesagainst E. chaffeensis but E. canis, to generate a score for E.chaffeensis. The assay results, optionally the scores, are compared toeach other to determine whether the infecting species is E. canis or E.chaffeensis. In some embodiments, if the score for E. canis is higherthan the score for E. chaffeensis, the sample is classified as infectedwith E. canis but not E. chaffeensis. In other embodiments, if the scorefor E. chaffeensis is higher than the score for E. canis, the sample isclassified as infected with E. chaffeensis but not E. canis. In someembodiments, if the two scores are identical, the sample is classifiedas infected with both E. chaffeensis and E. canis or as undetermined.

In certain embodiments, the sample used in the methods is from a wildanimal (e.g., a deer or rodent, such as a mouse, chipmunk, squirrel,etc.). In other embodiments, the sample is from a lab animal (e.g., amouse, rat, guinea pig, rabbit, monkey, primate, etc.). In otherembodiments, the sample is from a domesticated or feral animal (e.g., adog, a cat, a horse). In still other embodiments, the sample is from ahuman. In other embodiments, the sample is from a canine or felinesubject. In some embodiments, the sample is a bodily fluid. Inparticular embodiments, the sample is a blood, serum, plasma, cerebralspinal fluid, mucus, urine, or saliva sample. In certain embodiments,the sample is a whole blood sample. In other embodiments, the sample isa tissue (e.g., a tissue homogenate), tissue extract, or a cell lysate.

Much of the preceding discussion is directed to the detection ofantibodies against pathogenic Ehrlichia. However, it is to be understoodthat the discussion also applies to the detection of primed T-cells,either in vitro or in vivo.

It is expected that a cell-mediated immune response (e.g., a T-helperresponse) is generated, since IgG is produced. It is therefore expectedthat it will be possible to determine the immunological reactivitybetween primed T-cells and a population of peptides as described herein.In vitro this can be done by incubating T-cells isolated from thesubject with the population of peptides and measuring theimmunoreactivity, e.g., by measuring subsequent T-cell proliferation orby measuring release of cytokines from the T-cells, such as IFN-γ. Thesemethods are well-known in the art.

When a method of the invention is carried out in vivo, any of a varietyof conventional assays can be used. For example, one can perform anassay in the form of a skin test, e.g., by intradermally injecting, inthe subject, a population of peptides as described herein. A positiveskin reaction at the location of injection indicates that the subjecthas been exposed to and infected with the Ehrlichia species that thepopulation of peptides is specific to. The species of Ehrlichiainfecting the subject can be identified using the method of inventionwith the populations of peptides as described herein. This or other invivo tests rely on the detection of a T-cell response in the subject.

Certain embodiments of the method further comprise reporting detectionresults. The reporting can be done electronically, in writing, orverbally. It can be done via a machine such as a computer.

In yet another aspect, the invention provides kits. In some embodiments,the kits comprise at least one population of isolated peptides asdescribed herein. In particular embodiments, a kit comprises at leasttwo or three different populations of peptides. In some embodiments, akit comprises a first, second, and/or third populations of peptides asdescribed herein. In certain embodiments, the kits further comprise aninstruction.

In some embodiments, the kit is a kit for detecting antibodies that bindto Ehrlichia antigens and/or identifying the species of Ehrlichiainfecting a subject, if present.

In certain embodiments, the kit comprises:

-   -   a first population of isolated peptides as described herein;    -   a second population of isolated peptides as described herein;    -   a third population of isolated peptides as described herein; and    -   an instruction for using the first, second, and third        populations of peptides to identify the species of Ehrlichia in        a biological sample, if present.

In particular embodiments of the kits, the first population of isolatedpeptides is capable of specifically binding to antibodies againstantigens from multiple species of Ehrlichia including E. canis, E.chaffeensis, and E. ewingii. In other embodiments, the first populationof isolated peptides comprises at least three different peptides, eachcomprising a sequence of SEQ ID NO: 1 or a fragment thereof as describedherein. Specific examples of the peptide sequences with SEQ ID NO: 1that can be used in the kits have been described above, e.g., those withspecific amino acids at locations that can have various amino acids.Some specific examples are SEQ ID NOs: 4-51. Fragments of SEQ ID NO: 1that can be used in the kits have also been described above.

In other particular embodiments of the kits, the second population ofisolated peptides is capable of specifically or preferentially bindingto antibodies against antigens from E. ewingii, but not to or notpreferentially to antibodies against antigens from E. canis or E.chaffeensis. In other embodiments, the second population of isolatedpeptides comprises at least three different peptides, each comprising asequence of SEQ ID NO: 2 or a fragment thereof as described herein.Specific examples of the peptide sequences with SEQ ID NO: 2 that can beused in the kits have been described above, e.g., those with specificamino acids at locations that can have various amino acids. Somespecific examples are SEQ ID NOs: 52-66. Fragments of SEQ ID NO: 2 thatcan be used in the kits have also been described above.

In yet other embodiments of the kits, the third population of isolatedpeptides is capable of specifically or preferentially binding toantibodies against antigens from E. canis and E. chaffeensis, but not toor not preferentially to antibodies against antigens from E. ewingii. Inother embodiments, the third population of isolated peptides comprisesat least two or three different peptides, each comprising a sequence ofSEQ ID NO: 3 or a fragment thereof as described herein. Specificexamples of the peptide sequences with SEQ ID NO: 3 that can be used inthe kits have been described above, e.g., those with specific aminoacids at locations that can have various amino acids. Some specificexamples are SEQ ID NOs: 67-120. Fragments of SEQ ID NO: 3 that can beused in the kits have also been described above.

In certain embodiments of the kits, the peptide populations are attachedto or immobilized on a solid support. In some embodiments, the peptidepopulations are attached to or immobilized on a solid support through ametallic nanolayer (e.g., cadmium, zinc, mercury, gold, silver, copper,or platinum nanolayer). In certain embodiments, the solid support is abead (e.g., a colloidal particle or a metallic nanoparticle ornanoshell), a flow path in a lateral flow immunoassay device, a flowpath in an analytical or centrifugal rotor, a tube or a well (e.g., in aplate), or a sensor (e.g., an electrochemical, optical, oronto-electronic sensor).

Reagents for particular types of assays can also be provided in kits ofthe invention. Thus, the kits can include a population of beads (e.g.,suitable for an agglutination assay or a lateral flow assay), or a plate(e.g., a plate suitable for an ELISA assay). In other embodiments, thekits comprise a device, such as a lateral flow immunoassay device, ananalytical or centrifugal rotor, a Western blot, a dot blot, a slotblot, or an electrochemical, optical, or opto-electronic sensor. Thepopulation of beads, the plate, and the devices are useful forperforming an immunoassay. For example, they can be useful for detectingformation of an antibody-peptide complex comprising an antibody from asample and a peptide of the invention. In certain embodiments, apeptide, a mixture of different peptides (i.e. population of peptides)of the invention, or a peptide composition of the invention is attachedto or immobilized on the beads, the plate, or the device.

In addition, the kits can include various diluents and buffers, labeledconjugates or other agents for the detection of specifically boundantigens or antibodies (e.g. labeling reagents), and othersignal-generating reagents, such as enzyme substrates, cofactors andchromogens. In some embodiments, the kit comprises an anti-human,anti-canine, or anti-feline IgG/IgM antibody conjugated to a detectablelabel (e.g., a metallic nanoparticle, metallic nanoshell, metallicnanolayer, fluorophore, quantum dot, colored latex particle, or enzyme)as a labeling reagent. In other embodiments, the kit comprises proteinA, protein G, protein A/G fusion proteins, protein L, or combinationsthereof conjugated to a detectable label (e.g., a metallic nanoparticle,metallic nanoshell, metallic nanolayer, fluorophore, colored latexparticle, or enzyme) as a labeling reagent. An exemplary protein A/Gfusion protein combines four Fc-binding domains from protein A with twofrom protein G. See, e.g., Sikkema, J. W. D., Amer. Biotech. Lab, 7:42,1989 and Eliasson et al., J. Biol. Chem. 263, 4323-4327, 1988, bothwhich are hereby incorporated by reference in their entireties.

Other components of a kit can easily be determined by one of skill inthe art. Such components may include coating reagents, polyclonal ormonoclonal capture antibodies specific for a population of peptides asdescribed herein, purified or semi-purified extracts of these antigensas standards, monoclonal antibody detector antibodies, an anti-mouse,anti-dog, anti-cat, anti-chicken, or anti-human antibody conjugated to adetectable label, indicator charts for colorimetric comparisons,disposable gloves, decontamination instructions, applicator sticks orcontainers, a sample preparatory cup, etc. In one embodiment, a kitcomprises buffers or other reagents appropriate for constituting areaction medium allowing the formation of a peptide-antibody complex.

In certain embodiments, the kits comprise an instruction indicating howto use the first, second, and/or third populations of isolated peptidesas described herein to detect an antibody to an Ehrlichia antigen and/orto identify the species of Ehrlichia infecting a subject, if present. Incertain embodiments, the kits comprise an instruction indicating how touse a population of beads, a plate, or a device (e.g., comprising apeptide or a population of peptides of the invention) to detect anantibody to one or more Ehrlichia antigens and/or to identify thespecies of Ehrlichia. In particular embodiments, the instructioncomprises directions to identify the species of Ehrlichia infecting asubject, if present, according to the methods described herein. Incertain embodiments, the instruction comprises directions to contact abiological sample with the first, second, and third populations ofpeptides separately. In particular embodiments, the instructioncomprises directions to contact a biological sample with the first,second, and third populations of peptides sequentially.

Such kits provide a convenient, efficient way for a clinical laboratoryto diagnose infection by a pathogenic Ehrlichia and/or identifying thespecies of Ehrlichia infecting a subject.

In another aspect, the invention provides compositions useful foridentifying the species of Ehrlichia infecting a subject, if present. Insome embodiments, the composition comprises at least one population ofisolated peptides as described herein. In certain embodiments, theinvention provides a combination of compositions comprising the first,second, and third populations of peptides, respectively.

In another aspect, the invention provides devices useful for identifyingthe species of Ehrlichia infecting a subject, if present. In someembodiments, the device comprises at least one population of isolatedpeptides as defined above. In certain embodiments, the device comprisesthe first, second, and third populations of peptides.

In certain embodiments, the devices are useful for performing animmunoassay. For example, in certain embodiments, the device is alateral flow immunoassay device. In some embodiments, the device is aslide comprised of a plurality of beads to which a peptide or populationof peptides is attached. In other embodiments, the device is ananalytical or centrifugal rotor. In other embodiments, the device is adot blot, slot blot, or Western blot. In other embodiments, the deviceis a tube or a well, e.g., in a plate suitable for an ELISA assay. Instill other embodiments, the device is an electrochemical sensor, anoptical sensor, an opto-electronic sensor, an X-ray film,chemi-luminescence imager or a photon detection equipment.

The methods, kits, compositions, and devices of the invention offer anumber of advantages. For example, they allow for simple, inexpensive,rapid, sensitive and accurate detection of antibodies against Ehrlichiaand identification of the species of Ehrlichia infecting a subject, ifpresent. They also avoid serologic cross-reactivity with otherconditions with similar symptoms. This allows for an accurate diagnosisof the bacteria and species, thereby facilitates timely and appropriatetreatment that may be needed for the particular species of Ehrlichia.

The following examples illustrate various aspects of the invention. Theexamples should, of course, be understood to be merely illustrative ofonly certain embodiments of the invention and not to constitutelimitations upon the scope of the invention.

EXAMPLES Example 1—Experimental Infection of Dogs with Ehrlichia andDetection of Species-Specific Anti-Ehrlichia Antibodies with ELISA

This example shows that antibodies specific to particular Ehrlichiaspecies were generated and found reactive to the populations of peptidesas described herein.

A number of dogs were experimentally infected with E. canis, E.chaffeensis or E. ewingii (four dogs for each Ehrlichia species) for thepurpose of studying the course of pathological changes and antibodyproduction. The animals were infected using cultures of E. canis and E.chaffeensis, respectively, and blood stabilates of E. ewingii (E.ewingii has not been successfully cultured, and thus no slides forconducting IFA are currently available for this species.) Plasma sampleswere drawn from the infected dogs at various time points to generatedthe “dog plasma samples”. Although all of the infected animals showedthe presence of bacterial DNA by PCR, in the time period allowed for thestudy, only one of the E. chaffeensis-infected dogs and two of the E.canis-infected dogs showed the presence of anti-bacterial antibodies asdetermined by reactivity with SNAP 4DX Plus™ (manufactured by IDEXXLaboratories, Inc., which detects antibodies against E. canis, E.ewingii and E. chaffeensis). All the dog plasma samples from theinfection study found positive on SNAP 4Dx Plus™ were also positive inthe ELISA assays performed according to the method described below,using the first population of peptides as described below (ECHEW1).

Three different populations of peptides were synthesized using standardsynthesis procedures. Each peptide in the first population of peptides(ECHEW1) contained a sequence of SEQ ID NO: 1, which comprises achimeric peptide encompassing two different sequences that bindantibodies elicited to the following Ehrlichia antigens: msp4, p30 orp30-1 from canis/chaffeensis and 28 kD from ewingii. The ECHEW1population of peptides specifically binds to antibodies elicited bymultiple Ehrlichia spp. (E. canis, E. chaffeensis, and E. ewingii). Eachpeptide in the second population of peptides (EE13) contained a sequenceof SEQ ID NO: 2. The EE13 population of peptides specifically binds toantibodies elicited primarily by E. ewingii with some lowcross-reactivity to E. canis and E. chaffeensis. Each peptide in thethird population of peptides (EE12EW1) contained a sequence of SEQ IDNO: 3. The EE12EW1 population of peptides specifically binds toantibodies elicited primarily by E. canis and E. chaffeensis with somelow cross-reactivity to E. ewingii.

ELISA Method

-   1. Coating Antigen    -   1.1. The desired number of wells in 96-well plates (Thermo        Scientific Nunc™ MaxiSorp” Microplates) were coated with 1-20        μg/mL of Abaxis Ehrlichia antigen population ECHEW1, EE12EW1, or        EE13, each conjugated to BSA and diluted in 0.1 M sodium        carbonate/bicarbonate buffer (pH 9-9.4). Coating was performed        by adding 0.1 mL of the antigen to each well and incubating the        plate on a micro plate shaker at 250-300 rpm at room temperature        for approximately one hour.    -   1.2. The coating solution was removed, followed by dabbing the        plates on paper towels to eliminate any hanging droplets. 0.3 mL        of deionized water was added to each well, and the plates were        shaken at 250-300 rpm for 5 minutes. The liquid was removed as        above.    -   1.3. The wash step as in 1.2 was repeated twice.-   2. Blocking of the Plate    -   2.1. The coated plate wells were blocked by treating with the        blocking solution consisting of 30 g of non-fat milk in 100 mL        of deionized water. Each well was filled with 0.3 ml of blocking        solution and the plates were placed on a shaker at 250-300 rpm        for approximately one hour.    -   2.2. Blocking solution was removed and the plate was dabbed on a        paper towel to remove hanging droplets.-   3. Sample/Calibrator Incubation    -   3.1 Anti-Ehrlichia antibody calibrators were generated from        canine plasma by making a pool of high titer plasma samples        against known species. Species was determined by SNAP 4DX Plus™        and SNAP 3DX™ (manufactured by IDEXX, which detects antibodies        against E. canis and E. chaffeensis, but not E. ewingii)        differential testings and IFA. The pool was then assigned an        arbitrary score and diluted to various levels in a negative        canine plasma diluent. The score scaled linearly with the        dilution: for example, if a sample with score 40 was diluted 2        fold the resulting score would be 20. A set of five Ehrlichia        calibrators was run on each plate for the Ehrlichia ELISA. One        set was comprised of plasma samples that were positive to E.        canis, E. chaffeensis and E. ewingii, and was used with an        ECHEW1-coated plate. One set was comprised of anti-E. canis/E.        chaffeensis-positive samples, using samples that show close        titers in the IFA for canis and chaffeensis, respectively, and        was used with an ECHEW1-coated plate and a EE12EW1-coated plate.        Another set was comprised of anti-E. ewingii-positive samples        and was used with a EE13-coated plate. Each dog plasma sample or        calibrator was diluted 250-fold in the blocking solution.        Aliquots of 0.1 mL of each of the diluted calibrators and the        dog plasma samples were added to the wells and plates were        placed on the shaker at 250-300 rpm for one hour. Both the        calibrators and dog plasma samples were run in duplicate and the        results reported are the average of the two readings.    -   3.2. The sample solution was removed and the plate was washed in        the washing buffer containing 50 mM Trizma base (Sigma-Aldrich        T1503) and 0.05% CHAPS detergent (pH 8.0) (Sigma-Aldrich C3023).        The washing step was carried out by adding 0.3 mL of the washing        buffer and shaking the plate at 250-300 rpm for 5 minutes. The        washing solution was removed by inverting the plate and then        dabbing on a paper towel to eliminate any hanging droplets.    -   3.3 The above washing step was repeated twice.-   4. Conjugate Incubation    -   4.1. Protein A-HRP conjugate (Bio-Rad 170-6522) was diluted        8000-fold in the blocking solution (described in 2.1 above) and        0.1 mL of the diluted conjugate was added to each well. The        plates were then incubated with shaking at 250-300 rpm at room        temperature for approximately one hour.    -   4.2. The conjugate was removed and the plates were dabbed on a        paper towel to remove hanging droplets. The plates were washed        thrice as described above in 3.2 and 3.3. Finally, the plates        were washed with 0.3 mL of distilled water per well.    -   4.3. The bound conjugate was assayed by adding 0.1 mL of the        substrate TMB solution (Millipore ES022). The substrate was        allowed to react for 10 min at room temperature before OD 650 nm        readings were taken on a plate reader (Spectramax 340 PC.).

Plasma samples from the infected dogs were drawn at several time pointsand assayed with the Ehrlichia ELISA method as described above usingECHEW1, EE13, and EE12EW1, respectively. The results are shown in FIG.2. These results show the reactivity of ECHEW1 and EE12EW1 withantibodies produced in response to E. canis and E. chaffeensis. Theantibodies produced in response to E. canis did not react with E.ewingii-specific peptide population EE13. A very slight cross-reactivityof the 42 day-post-infection sample from the E. chaffeensis-infecteddogs with EE13 was noted.

Example 2—Detection of Presence of and Species-Specific Antibodies fromAdditional Known Anti-Ehrlichia-Positive or Negative Samples Using thePopulations of Peptides

This example shows the detection of the presence of anti-Ehrlichiaantibodies and, if present, the species-specific antibodies fromadditional samples that were identified by reference methods to beanti-Ehrlichia-positive or negative, using the ECHEW1, EE13, and EE12EW1populations of peptides in ELISA. It shows that the ELISA results agreewith reference method results.

Each peptide in the three populations, ECHEW1, EE13, and EE12EW1, waslinked separately to the carrier protein bovine serum albumin (BSA)using thio-ether chemistry. The resulting BSA-peptide conjugates wereused as capture entities in 96-well ELISA plates to create threeseparate ELISA assays (one population of peptides per plate). The plateswere then blocked to prevent undesirable non-specific binding.

A total of 224 anti-Ehrlichia-positive samples (dog plasma samplespositive to E. Canis, E. chaffeensis, or E. ewingii as determined by IFAand SNAP 4DX Plus™/SNAP 3Dx™) and 264 anti-Ehrlichia-negative samples(244 dog plasma samples and 20 dog whole blood samples negative to E.Canis, E. chaffeensis, and E. ewingii as determined by the samereference methods) were incubated with the immobilized peptidepopulations in each of the three ELISA plates. After one hourincubation, the unreacted materials were removed by washing the microwells. The specifically captured dog IgG or IgM were detected byreaction with HRP-labeled Protein A. HRP was assayed using a commercialTMB substrate. The optical density of each well was read at 650 nm witha plate reader. A summary of the results separated by “Sample Status”,from IFA and SNAP tests, is shown in Table 1 below.

TABLE 1 ELISA Results of Known Ehrlichia-Positive or Negative SamplesELISA Result² ECHEW1- Positive with ECHEW1- EE12EW1 > Positive withECHEW1- Sample Status¹ EE13 EE13 > EE12EW1 Negative Total E. canis 50 01 51 E. chaffeensis 49 4 4 57 E. ewingii 2 80 10 92 Positive, species 231 0 24 indeterminate Negative 2 3 259 264 ¹The Sample Status wasdetermined from the results of IFA and SNAP tests. ²An ELISA Result forECHEW1 was classified as “positive” if it had a score ≧3 or “negative”if it had a score <3.

Of the 224 anti-Ehrlichia-positive samples (determined by IFA and SNAPtests), 209 were identified positive by our ELISA assay using thepeptide population ECHEW1. Thus, the percent sensitivity of the ELISAECHEW1 was 93.3%. Of the 264 anti-Ehrlichia-negative samples, 259 wereidentified negative by our ELISA assay. Thus, the percent specificity ofthe ELISA ECHEW1 was 98.1%. Furthermore, of the 108 samples that wereclassified as anti-E. canis-specific or anti-E. chaffeensis-specific byIFA and SNAP tests, 99 (“ECHEW1-Positive with EE12EW1>EE13”) werecorrectly identified by our ELISA detection process. Of the 92 samplesthat were classified as anti-E. ewingii-specific by IFA and SNAP tests,80 (“ECHEW1-Positive with EE13>EE12EW1”) were identified by our ELISAdetection process. Therefore, our ELISA methods are in good agreementwith the reference methods.

In addition, of the 25 anti-Ehrlichia-positive samples whose speciesinformation could not be determined by IFA or SNAP assays, our ELISAidentified them to be either E. Canis/E. chaffeensis specific (if theEE12EW1 score was greater than the EE13 score) or E. ewingii specific(if the EE13 score was greater than the EE12EW1 score), with fairly highconfidence.

In some embodiments, lateral flow immunoassays can be used in place ofthe ELISA assays in methods described above. Therefore, other assayformats employing the populations of peptides as described herein can beused in the methods of the invention to identify Ehrlichia species.

Example 3—Generation of Standard Curves and Identification of ThreeUnknown Samples

This example demonstrates in detail how standard curves for the threepopulations of isolated peptides, ECHEW1, EE13, and EE12EW1, could begenerated, as well as how three unknown samples were classifiedaccording to the methods of the invention.

An ELISA assay was performed according to the method described inExample 1. In particular, a set of five Ehrlichia calibrators generatedfrom known canine plasma samples as described in Example 1 was run oneach plate for the Ehrlichia ELISA. One set was comprised of E. canis/E.chaffeensis positive samples and was used with an ECHEW1-coated plateand an EE12EW1-coated plate. Another set was comprised of E. ewingiipositive samples and was used with an EE13-coated plate.

Each of three unknown canine plasma samples was diluted 250, 500 and1000-fold in the blocking solution. Aliquots of 0.1 mL of each of thediluted calibrators and the unknown samples were then added to the wellsand plates were placed on the shaker at 250-300 rpm for one hour.

Both the calibrators and unknown samples were run in duplicate and theresults reported are the average of the two readings.

Data Analysis

A standard curve for each population of peptides was prepared by usingthe respective ELISA calibrators with scores (ECHEW1 Score for allspecies, EE12EW1 Score for canis and/or chaffeensis, and EE13 Score forewingii) on the x-axis and optical density (OD) on the y axis. TheEhrlichia scores of the unknown samples were interpolated from thisstandard curve. The ECHEW1 Score, EE12EW1 Score, or EE13 Score for anunknown sample was determined by using the OD from a dilution that fallswithin the calibration curve.

Results

The ELISA results (OD 650 nm readings) of the calibrators are shown inTable 2:

TABLE 2 Standard Curves (Assigned Scores and OD Readings of Calibrators)OD 650 nm OD 650 nm OD 650 nm Score ECHEW1 EE12EW1 EE13 0 0.00 0.00 0.0010 0.04 0.03 0.06 40 0.40 0.24 0.13 80 0.72 0.43 0.28 120 1.03 0.68 0.83

Standard curves were calculated as follows:ECHEW1: OD=0.0088(ECHEW1 Score)+0.0027EE12EW1: OD=0.0055(EE12EW1 Score)+0.005EE13: OD=0.0069(EE13 Score)+0.0029

The ELISA results of the unknown samples are shown in Table 3:

TABLE 3 ELISA OD Readings of Unknown Samples OD 650 nm OD 650 nm OD 650nm Sample Name ECHEW1 EE12EW1 EE13 Unknown 1 0.42 0.03 0.34 Unknown 20.48 0.31 0.01 Unknown 3 0.0003 0.012 0.002

Scores of the unknown samples were calculated by the following formula:(SCORE)=(OD−B)/A

-   -   Where B is the intercept of the standard curve and A is the        slope.    -   For each score the OD and the constants used come from the        peptide population in question.

The scores calculated for each unknown sample are as follows:

-   -   1.) Unknown 1    -   (ECHEW1 Score)=(0.42−0.0027)/0.0088=47    -   (EE12EW1 Score)=(0.03−0.005)/0.0055=5    -   (EE13 Score)=(0.34−0.0029)/0.0069=49

The ECHEW1 Score was used to determine if the sample is positive ornegative for infection with any species from E. canis, E. chaffeensis,and E. ewingii. Then the EE13 Score was compared to the EE12EW1 Score todetermine the species of the infection. In this case, for Unknown 1,ECHEW1 Score is positive, and EE13 Score>>EE12EW1 Score, so the sampleis positive for E. ewingii.

-   -   2.) Unknown 2    -   (ECHEW1 Score)=(0.48−0.0027)/0.0088=54    -   (EE12EW1 Score)=(0.31−0.005)/0.0055=55    -   (EE13 Score)=(0.01−0.0029)/0.0069=1    -   EE12EW1 Score>>EE13 Score, so the sample is positive for E.        canis/E. chaffeensis.    -   3.) Unknown 3    -   (ECHEW1 Score)=(0.003−0.0027)/0.0088=0    -   (EE12EW1 Score)=(0.012−0.005)/0.0055=1    -   (EE13 Score)=(0.002−0.0029)/0.0069=0    -   All three scores are very low so the sample is negative for all        three Ehrlichia species.        Cutoff

The cutoff for the ELISA test method was calculated on the basis ofanalysis of 294 samples, 128 negatives and 166 positives. These sampleswere classified by use of SNAP 4Dx Plus and E. Canis and E. ChaffeensisIFA titers. The samples used in this study were any for which bothmethods agreed, i.e., both SNAP and IFA were Positive or both wereNegative. In this case whichever IFA titer was higher was the valueused. Each of these 294 samples was tested using ELISA assays accordingto the procedure described in Example 1, and an antibody level score wasassigned to each assay result for each sample. Positive and negativestatus for a sample run through this ELISA assay were determined on thebasis of ECHEW1 Score alone so all the calculations here were concerningthe ECHEW1 score for these samples.

The cutoff was set at three standard deviations above the negative mean.For this sample set that is:

-   -   Mean of Negative samples 0.37    -   Standard Deviation of Negative samples 0.82    -   Mean+3×{StDev} 2.84

In this Example all scores were rounded to the nearest integer so anysample with an ECHEW1 Score>=3 were considered a positive. At an ELISAscore of 3, one would expect 99.2% Specificity and 95.8% Sensitivity.

To the extent that any definitions in documents incorporated byreference are inconsistent with the definitions provided herein, thedefinitions provided herein are controlling. Although the invention hasbeen described with reference to the presently preferred embodiments, itshould be understood that various changes and modifications, as would beobvious to one skilled in the art, can be made without departing fromthe spirit of the invention. Accordingly, the invention is limited onlyby the following claims.

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

What is claimed:
 1. A kit comprising: a first population of isolatedpeptides comprising at least three different peptides, each comprising asequence of SEQ ID NO: 1; a second population of isolated peptidescomprising at least three different peptides, each comprising a sequenceof SEQ ID NO: 2; a third population of isolated peptides comprising atleast three different peptides, each comprising a sequence of SEQ ID NO:3; and an instruction for using the first, second, and third populationsof peptides to identify the species of Ehrlichia in a biological sample,if present.
 2. The kit of claim 1, further comprising one or morelabeling reagents.
 3. The kit of claim 1, wherein the first, second,and/or third populations of peptides are immobilized to a solid support.4. The kit of claim 1, wherein the kit comprises one or more antibodiesconjugated to a detectable label.
 5. The kit of claim 4, wherein thedetectable label is selected from the group consisting of metallicnanoparticles, metallic nanoshells, metallic nanolayers, fluorophores,quantum dots, colored latex particles, and enzymes.
 6. The kit of claim1, wherein the kit comprises Protein A, Protein G, Protein A/G fusionproteins, Protein L, or combinations thereof conjugated to a detectablelabel.
 7. The kit of claim 1, wherein: X₃₉ in SEQ ID NO: 1 is K; X₄₄ inSEQ ID NO: 1 is K or R; X₄₉ in SEQ ID NO: 1 is E or D; X₅₆ in SEQ ID NO:1 is K or Q; X₅₈ in SEQ ID NO: 1 is E or T; X₇ in SEQ ID NO: 2 is K; X₁₂in SEQ ID NO: 2 is K or R; X₁₇ in SEQ ID NO: 2 is E or D; X₂₄ in SEQ IDNO: 2 is K or Q; and/or X₂₆ in SEQ ID NO: 2 is E or T.
 8. The kit ofclaim 1, wherein the instruction for using the first, second, and thirdpopulations of peptides to identify the species of Ehrlichia in abiological sample comprises instruction to perform at least one of saiddetecting steps selected from: (i) performing an ELISA assay; (ii)running a lateral flow assay; (iii) performing an agglutination assay;(iv) performing a Western blot, slot blot, or dot blot assay; (v)performing a wavelength shift assay; (vi) running the sample through ananalytical or centrifugal rotor; or (vii) running a microarray assay.